Insight Into the Biological Activity of Hennosides—Glucosides Isolated from Lawsonia Inermis (Henna): Could They Be Regarded As Active Constituents Instead

plants Article Insight into the Biological Activity of Hennosides—Glucosides Isolated from Lawsonia inermis (henna): Could They Be Regarded as Active Constituents Instead Irina Maslovarić 1 , Vesna Ilić 1 , Ivana Drvenica 1 , Ana Stanˇcić 1, Slavko Mojsilović 1 , Tamara Kukolj 1, Diana Bugarski 1, Luciano Saso 2 and Marcello Nicoletti 3,* 1 Institute for Medical Research, University of Belgrade, Dr Subotića4, POB 39, 11129 Belgrade 102, Serbia; [email protected] (I.M.); [email protected] (V.I.); [email protected] (I.D.); [email protected] (A.S.); [email protected] (S.M.); [email protected] (T.K.); [email protected] (D.B.) 2 Department of Physiology and Pharmacology “Vittorio Erspamer”, Sapienza University of Rome, Square Aldo Moro, 5, 00185 Rome, Italy; [email protected] 3 Department of Environmental Biology, Sapienza University of Rome, Square Aldo Moro, 5, 00185 Rome, Italy * Correspondence: [email protected] Abstract: Henna is the current name of the dye prepared from the dry leaf powder of Lawsonia inermis (Lythraceae). Several studies have focused on the chemistry and pharmacology of the henna dyeing active compound, lawsone, obtained from the main constituents of leaves, hennosides, during the processing of plant material. However, knowledge regarding the biological activity of hennosides is largely lacking. In this paper, the redox activity of three hennoside isomers is reported. The pro- Citation: Maslovarić,I.; Ilić,V.; oxidative activity was confirmed by their ability to induce mild lysis of erythrocytes and to increase Drvenica, I.; Stanˇcić,A.; Mojsilović,S.; the level of methemoglobin at the concentration ≥ 500 µg/mL. The antioxidant activity of hennosides Kukolj, T.; Bugarski, D.; Saso, L.; (concentration ≥100 µg/mL) was determined by FRAP and ABTS assays. At concentration of Nicoletti, M. Insight into the 500 µg/mL, antioxidant activity of hennoside isomers was equivalent to 0.46 ± 0.08, 0.62 ± 0.28 and Biological Activity of 0.35 ± 0.03 mM FeSO4 × 7H2O, and 0.15 ± 0.01, 0.30 ± 0.01 and 0.09 ± 0.01 mM Trolox. Hennosides Hennosides—Glucosides Isolated at 100 µg/mL concentration did not influence viability of human breast cancer cell lines MDA231 from Lawsonia inermis (henna): Could and MCF-7 and primary human peripheral blood and periodontal ligament-mesenchymal stem cells, They Be Regarded as Active but produced a modest increase in concentration of antioxidants in the cell culture supernatants. Plants 10 Constituents Instead. 2021, , The evidenced antioxidant and pro-oxidant activities indicate their potential to act as redox balance 237. https://doi.org/10.3390/ regulator, which opens up the possibility of using hennosides in commercial phytomedicines. plants10020237 Keywords: Lawsonia inermis; Lythraceae; glucosides; redox activity Received: 11 December 2020 Accepted: 19 January 2021 Published: 26 January 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in Lawsonia inermis L. (syn. L. alba o L. spinosa, family Lythraceae), vulgarly known as published maps and institutional affil- henna, hennè, shudi, madurang, manghati, madayantika and goranti, is a perennial shrub, iations. well known from ancient times as a medicinal and dyeing plant [1]. Nowadays, because of its strong coloring properties, the use of dried henna leaf powder for hair coloring and the body-decorating process, “temporary tattoos”, also known as mehndi [2,3], on the skin has increased. In the US, direct application to the skin is not approved by the Food and Drug Copyright: © 2021 by the authors. Administration [4]. Conversely, it is authorized and widely available as a coloring agent Licensee MDPI, Basel, Switzerland. for hair across the globe, including in the US [5]. The coloring action of red henna is due to This article is an open access article lawsone, 2-hydroxy-1,4-naphtoquinone. Nonetheless, to obtain the dying effect of henna, distributed under the terms and the leaves must be treated until the principal active ingredient, lawsone, is able to react via conditions of the Creative Commons Michael reaction with keratin in the skin and hair, resulting in a permanent dye stain: Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ C9H5O2-C=O + 2HN-Keratin ! C9H5O2-C=N-Keratin + H2O 4.0/). Plants 2021, 10, 237. https://doi.org/10.3390/plants10020237 https://www.mdpi.com/journal/plants Plants 2021, 10, 237 2 of 15 There is evidence, however, that lawsone, an aglycone, is an artifact arising from the oxidative transformation of the primary glycosidic constituents during the processing of plant material to obtain the dye [6]. The relation between glucosides and aglycone is common in natural products chemistry. Therefore, a preliminary treatment is necessary to convert the hennosides (glucosides), present originally in the raw material, into an active herbal dying principle, lawsone [7]. Conversion of hennosides into the unique aglycone requires that henna powder be soaked in mildly acidified solution for 6 to 24 h before use. Hennosides are three isomer glucosides, where each of the hydroxyls, derived from the interconversion of the two keto-enol forms of the naphtoquinone structure, can be glycosylated, leading to hennoside isomerism. The aglycone, derived from their hydrolysis, is then converted by oxidation into lawsone, the dying active compound (Figure1)[ 7]. The henna drug content is usually referred to as the lawsone content, but as demonstrated [7], lawsone, as a free molecule, is absent in the raw material, and hennosides, precursors of henna, that are co-occurring in the activity with lawsone, could be regarded as active constituents instead [8] and serve to evaluate any biological activity of henna leaves exploited in traditional medicine in many countries. Figure 1. Conversion of hennosides (isomerglucosides) into lawsone (aglycone). In fact, although the cosmetic use of henna has obscured its medicinal importance, the use of the leaves is well present in all the cultures producing or using the plant. The Ebers Papyrus, dated to about 1550 B.C., is one of the oldest known scrolls and the main source of herbal knowledge of medicine in ancient Egypt [9]. Henna is reported as a pharmacologically important plant with significant in vitro and in vivo biological activities. Several pharmacological activities such as antibacterial, antioxidant, anti-inflammatory and anticancer properties have been documented, of which the antioxidant and antimicrobial activities have been most thoroughly investigated [1]. In the dried leaves, besides lawsone, other compounds present like, polyphenols and glucides are also co-occurring in the activity of henna [8]. However, the information about its most particular constituents is insufficient. In this paper, we evaluate the biological properties, redox, and cytotoxic activity of hennosides, designated A, B and C, respectively. Unlike lawsone, which is easily chemically produced, hennosides are difficult to obtain in pure form, and often they are referred to as lawsone. This study could be also of ethnopharmacological interest, to confirm or reconsider the use of henna in traditional medicine through the definition of hennoside activity, and it could serve as a valuable source of information for carrying out further studies on this plant in future. 2. Results 2.1. Antioxidant Activity of Hennosides When hennosides were dissolved in 0.9% NaCl, both FRAP (ferric ion reducing antioxidant power) and ABTS (2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assays showed antioxidant activity at hennoside concentration ≥ 100 µg/mL (Figure2, Table S1). The highest antioxidant activity was confirmed for hennoside B with both assays. When Plants 2021, 10, 237 3 of 15 hennosides were dissolved in complete cell culture Dulbecco’s Modified Eagle Medium containing 10% fetal calf serum (D’MEM/10%FCS), their antioxidant capacity could not be measured with ABTS assay. This result demonstrated the high antioxidant capacity of the cell culture medium, indicating masking of antioxidant activity of hennosides by cell culture medium. The FRAP assay showed the antioxidant activity of hennosides in the cell culture media at hennoside concentration ≥ 100 µg/mL (Figure2; Table S1). Figure 2. Antioxidant activity of hennosides in isotonic NaCl and Dulbecco’s Modified Eagle Medium supplemented with 10% fetal calf serum (D’MEM/FCS). Antioxidant activity measured by FRAP (a,c) and ABTS (b,d) assay. HA—hennoside A; HB—hennoside B; HC—hennoside C; NaCl—0.9% NaCl; D’MEM/FCS: D’MEM cell culture medium supplemented with 10%FCS; (a,b) Values represent the concentration of antioxidants diluted in 0.9% NaCl. (c,d) Values represent the concentration of antioxidants diluted in D’MEM + 10%FCS; Red lines indicate mean—1SD (D’MEM/FCS − SD) to mean + 1SD (D’MEM/FCS + SD) range of antioxidant concentration in D’MEM/FCS. 2.2. Effect of Hennosides on Erythrocyte lysis The extent of hemolysis was determined by measuring the absorbance (optical density) at 540 nm (OD540) of supernatants obtained after centrifugation of erythrocyte suspension incubated with hennosides. A mild hemolysis was recorded in the presence of hennosides at ≥500 µg/mL concentration (Figure3, Table S2). Furthermore, the results showed the lack of hemolytic effect of dimethyl sulfoxide (DMSO), used as a stock solution for hennosides, in the concentrations applied. Plants 2021, 10, 237 4 of 15 Figure 3. Hennoside-induced lysis of erythrocytes resuspended in (a) 0.9% NaCl and (b) 0.9% NaCl containing 10% FCS (NaCl/FCS). The hemolysis was followed based on the optical density at 540 nm (OD540) values of supernatants obtained after centrifugation of erythrocyte suspension. HA—hennoside A; HB—hennoside B; HC—hennoside C; NaCl—hennosides and erythrocytes diluted in 0.9% NaCl; NaCl/FCS—hennosides and erythrocytes diluted in 0.9% NaCl containing 10% FCS. Red lines indicate mean − 2SD (control − 2SD) to mean + 2SD (control + 2SD) range of control i.e., spontaneous lysis of erythrocytes incubated in 0.9% NaCl (a) and (b) 0.9% NaCl/FCS without hennosides.

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